1. Field of the Invention
The present invention relates to the microbiological industry, and specifically to a novel aldolase and methods for manufacturing 4-hydroxy-L-isoleucine or a salt thereof.
2. Brief Description of the Related Art
4-hydroxy-L-isoleucine is an amino acid which can be extracted and purified from fenugreek seeds (Trigonella foenum-graecum L. leguminosae). 4-hydroxy-L-isoleucine displays an insulinotropic activity, which is of great interest because its stimulating effect is clearly dependent on the plasma glucose concentration in the medium, as demonstrated both in isolated perfused rat pancreas and human pancreatic islets (Sauvaire, Y. et al, Diabetes, 47: 206-210, (1998)). Such dependency on glucose has not been confirmed with sulfonylureas (Drucker, D. J., Diabetes 47: 159-169, (1998)), which are the only insulinotropic drug currently used to treat type II diabetes [or non-insulin-dependent diabetes (NIDD) mellitus (NIDDM)]. As a consequence, hypoglycemia remains a common undesirable side effect of sulfonylurea treatment (Jackson, J., and Bessler, R. Drugs, 22: 211-245; 295-320, (1981); Jennings, A. et al. Diabetes Care, 12: 203-208, (1989)). Methods for improving glucose tolerance (Am. J. Physiol. Endocrinol., Vol. 287, E463-E471, 2004) are also known. This glucometabolism enhancement activity, and its potential application to pharmaceuticals and health foods, has been reported (Japanese Patent Application Laid-Open No. Hei 6-157302).
Due to its particular insulinotropic action, 4-hydroxy-L-isoleucine, which is only found in plants, might be considered as a novel secretagogue for the treatment of type II diabetes. This is because Type II diabetes is characterized by defective insulin secretion associated with various degrees of insulin resistance (Broca, C. et al, Am. J. Physiol. 277 (Endocrinol. Metab. 40): E617-E623, (1999)).
A method of oxidizing iron, ascorbic acid, 2-oxyglutaric acid, and oxygen-dependent isoleucine by utilizing dioxygenase activity in fenugreek extract has been reported as a method for manufacturing 4-hydroxy-L-isoleucine (Phytochemistry, Vol. 44, No. 4, pp. 563-566, 1997). However, this method is unsatisfactory as a method of manufacturing 4-hydroxy-L-isoleucine because the activity of the enzyme is inhibited by the substrate at isoleucine concentrations of 20 mM and above, the enzyme has not been identified, the enzyme is derived from plant extracts and cannot be readily obtained in large quantities, and the enzyme is unstable.
An efficient eight-step synthesis of optically pure (2S,3R,4S)-4-hydroxyisoleucine with 39% overall yield has been disclosed. The key steps of this synthesis involve the biotransformation of ethyl 2-methylacetoacetate to ethyl (2S,3S)-2-methyl-3-hydroxy-butanoate with Geotrichum candidum and an asymmetric Strecker synthesis (Wang, Q. et al, Eur. J. Org. Chem., 834-839 (2002)).
A short six-step chemoenzymatic synthesis of (2S,3R,4S)-4-hydroxyisoleucine with total control of stereochemistry, the last step being the enzymatic resolution by hydrolysis of a N-phenylacetyl lactone derivative using the commercially available penicillin acylase G immobilized on Eupergit C(E-PAC), has also been disclosed (Rolland-Fulcrand, V. et al, J. Org. Chem., 873-877 (2004)).
But currently, there have been no reports of producing 4-hydroxy-L-isoleucine by enzymatic transamination of 4-hydroxy-3-methyl-2-keto-pentanoic acid or by any other enzymatic conversion from any other starting materials.